Accurately measure cell concentrations by flow cytometry

The Invitrogen™ Attune™ NxT Flow Cytometer is a next-generation cytometer that uses traditional hydrodynamic focusing for low sample rates (10–20 μL/min, Figure 1A) and acoustic-assisted focusing for high sample rates (100–1,000 μL/min, Figure 1B). Acoustic-assisted hydrodynamic focusing is a revolutionary technology that precisely align cells using ultrasonic radiation pressure (>2 MHz) to transport particles into the center of the sample stream. This prefocused stream is then injected into the sheath stream, which supplies additional hydrodynamic pressure to the sample prior to interrogation with one or more lasers (Figure 1B). The combination of these two forces results in a narrow particle stream and uniform laser illumination, regardless of the sample input rate.

In cytometers that rely solely on hydrodynamic focusing, the sample is spread across a wider core stream as the flow rate increases, which results in less uniform laser illumination and broader population distributions (Figure 1B). To obtain optimal data with the lowest signal variability, a conventional flow cytometer must be run at the lowest sample rate, typically 10–20 μL/min; higher sample rates produce greater variability and less precise measurements. With acoustic-assisted hydrodynamic focusing, the Attune NxT cytometer avoids this compromise between data quality and sample rates by uncoupling cell alignment from sheath flow.

Another important advantage of the Attune NxT cytometer over traditional flow cytometers is its unique volumetric sample and sheath fluid delivery system. Samples are introduced to the Attune NxT cytometer with syringes, producing accurate measurements of the volumes of acquired samples, which in turn leads to accurate calculation of cell concentrations (conveniently displayed as events/μL by the Attune NxT software). In contrast, conventional hydrodynamic focusing systems do not measure the volume of the acquired sample. Instead, counting beads must be added to calibrate the acquired volumes before calculating cell concentrations, which can lead to the introduction of errors from pipetting, gating, and extra calculations. Here we present two experiments where the Attune NxT cytometer’s increased sampling rate and precise sample volume measurements produce highly accurate cell concentration data in dilute samples.

Measure the concentration of lymphocyte subpopulations

Human lymphocyte subpopulations were identified and their concentrations measured using a 6-color antibody panel for the common lymphocyte phenotypic markers CD3, CD4, CD8, CD19, CD45, and CD56 to identify helper T cells, suppressor T cells, B cells, and natural killer (NK) cells. The high sampling rate of the Attune NxT cytometer precluded the need to wash the whole blood during the protocol, minimizing cell loss. The choice of excitation channels provided by the Attune NxT cytometer equipped with 4 lasers (405, 488, 561, and 637 nm) permitted the selection of fluorescent CD-specific antibodies that did not display significant spectral overlap, eliminating color compensation and simplifying the workflow. Figure 2 shows the scatter plots and cell concentrations for all lymphocyte subpopulations. The accuracy and precision of the cell concentrations obtained in this experiment were verified by running the sample at three different flow rates (100, 200, and 500 μL/min) (Figure 3).

Figure 2. Lymphocyte subset analysis on the Attune NxT Flow Cytometer. A 100 μL aliquot of normal human whole blood was labeled with fluorophore-conjugated antibodies against CD surface markers, followed by red blood cell lysis using 2 mL of High-Yield Lyse Fixative-Free Lysing Solution, resulting in a 1:21 dilution of the blood. The sample was acquired on the 4-laser Attune™ NxT Flow Cytometer. (A) Lymphocytes are identified on a density plot of CD45 vs. side scatter with an oval gate around the lymphocyte (CD45+) population. (B) Cells in the lymphocyte gate are displayed on a density plot of CD3 vs. side scatter. Rectangle gates surround the CD3+ T cell and CD3– B and natural killer (NK) cell populations. (C) Cells in the CD3+ gate are then displayed on a density plot of CD4 vs. CD8 to quantify CD4+ helper T cells (CD4+ CD3+ CD45+) and CD8+ cytotoxic T cells (CD8+ CD3+ CD45+). (D) CD3– cells are displayed on a density plot of CD56 vs. CD19 to distinguish CD56+ NK cells from CD19+ B cells. The statistics table shows the gating and measured concentrations (cells per μL). The product list at the end of this article shows the antibody conjugates used.

Figure 3. Replicate samples collected at three flow rates on the Attune NxT Flow Cytometer. In the experiment described in Figure 2, cell concentrations were measured using three different flow rates: 100, 200, and 500 μL/min. The Attune™ NxT Flow Cytometer provides similar concentration measurements for each lymphocyte subpopulation, regardless of the flow rate. Each bar represents the mean cells/μL ± standard deviation of three samples run at each indicated flow rate for each population.

Analyze bacteria in wastewater

A sample of municipal wastewater was labeled with the Invitrogen™ LIVE/DEAD™ BacLight™ Bacterial Viability Kit to identify live and dead bacteria. On traditional flow cytometers, very dilute samples can take a long time to acquire due to slower flow rates. This sample (3 mL) was analyzed on the Attune NxT cytometer at a flow rate of 1 mL/min, which allowed quick analysis of the sample and accurate detection of very small quantities of bacteria. Moreover, we determined the concentrations of the live and dead bacteria without using reference counting beads

Figure 4 shows the two-parameter dot plot (propidium iodide vs. SYTO™ 9 fluorescence), with the live (green) and dead (red) bacterial populations well separated; the statistics table displays the concentration measurements for the labeled bacteria. Wastewater may also include small eukaryotes and types of bacteria that are potentially viable but nonculturable, each of which may also be labeled with the dyes; the grey dots represent debris found in the wastewater.

Figure 4. Analysis of bacteria in treated municipal wastewater on the Attune NxT Flow Cytometer. A sample of treated municipal wastewater was labeled with a 1:1 mixture of the cell-permeant, green-fluorescent SYTO™ 9 and cell-impermeant, red-fluorescent propidium iodide (PI) stains (components of the LIVE/DEAD™ BacLight™ Bacterial Viability Kit), incubated for 15 min protected from light, and then acquired on the Attune™ NxT Flow Cytometer at a flow rate of 1 mL/min with a forward scatter threshold. The blue 488 nm laser was used with a 530/30 nm bandpass emission filter for SYTO 9 detection in BL1, and a 695/40 nm emission filter for PI detection in BL3. SYTO 9–stained live (green) and PI-stained dead (red) populations of bacteria are demonstrated in a dual-parameter dot plot of SYTO 9 vs. PI fluorescence. Grey dots represent debris present in the wastewater sample. The statistics table shows the concentration of live (1 cell/μL) and dead (3 cells/μL) bacteria in the acquired sample.

Obtain high sample rates and accurate cell concentrations

With its high sampling rates and ability to accurately measure sample volume, the Attune NxT Flow Cytometer not only permits more streamlined workflows but also improves the accuracy of cell concentration measurements.